細胞遊走能・浸潤能

Cell migration across a scratch assay
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スクラッチアッセイは、スクラッチ処理を施した領域への細胞の移動を測定する in vitro アッセイです。がんや創傷治癒研究分野における細胞増殖や遊走評価に頻繁に用いられます。細胞の増殖や遊走の追跡は、その転移能を定量化する有効な手法の一つで、とりわけ乳がんの転移を抑制する化合物候補の評価には有用です。

スクラッチアッセイにより、比較的簡単・ローコストで細胞の遊走を追跡することができます。Axion BioSystems の Maestro シリーズでは、マルチウェルフォーマットの環境下で、ラベルフリー・リアルタイムにスクラッチアッセイを行うことが可能です。遊走の速さは細胞や薬剤のタイプ、或いは実験プロトコルによっても異なるため、これらのアッセイには繰り返しや多くの時間を要します。Maestro なら一度のアッセイで異なる条件下での変化を経時的に測定することが可能です。

 

スクラッチアッセイによる細胞遊走能の検証

トリプルネガティブ乳がんは、他の乳がんと比較して進行が早く、予後も不良です。また、転移や再発のリスクも高い一方で、その治療法は限定されています。

in vitro スクラッチアッセイにより、がん細胞の浸潤や転移能、また転移抑制化合物候補の薬効評価が可能です。本事例では、細胞インピーダンスの変化により、2種類の乳がん細胞株の遊走能を比較しています。

Scratch through confluent cells on plate

(A) プレート上でコンフルになった細胞に施されたスクラッチ処理(オレンジ色)

Cells migrating across scratch

(B) スクラッチ領域への細胞遊走(施術から24時間後の様子)

 

Cell migration continuous monitoring for 72 hours
Cell migration after scratch shows a drop in Impedance
After the scratch the impedance returned as the cells regrew
Impedance showed a lack of regrowth due to the addition of migration inhibiting coatings

 

MCF-7 (ホルモン受容体陽性乳がん細胞株)とHCC1806 (トリプルネガティブ乳がん細胞株)の2種類のがん細胞株を CytoView-Z プレート上に播種し、Maestro Z を用いて連続してインピーダンスを測定した。72時間後、低血清濃度の培地に交換し、更に24時間後にスクラッチ処理を施した。1部 well にはシトラスペクチン (MCP)を投与し、以降72時間以上にわたりインピーダンスを測定した。

(C) 細胞播種後72時間(スクラッチ施術前)のインピーダンスの変化。両細胞にて連続した上昇(細胞の増殖)が見られた。 (D, E) スクラッチ処理直後、両細胞において急激なインピーダンスの減少が得られた。HCC1806細胞は徐々に遊走しスクラッチ領域のほぼ全てを覆いつくした。また、インピーダンス(D: オレンジ色)は徐々に回復し、クラッチ非施術の細胞と同等レベルとなった。一方、MCF-7細胞はスクラッチ以降72時間に渡って殆ど遊走は見られず、そのインピーダンス(E:水色)に顕著な回復は得られなかった。(F) スクラッチ処理後、HCC1806にシトラスペクチン (MCP或いはPrctraSol-C) 投与したところ、インピーダンスの上昇が抑制され、遊走抑制効果が示唆された。

Cell Migration assay protocol or steps

Maestroによる、インピーダンスアッセイはとても簡単です。事前コーティングされた CytoView-Z プレート上に細胞を播種します (Hour 0)。Maestroシステムにプレートを搭載すると同時に温度・CO₂ 濃度制御とインピーダンス測定が開始されます。細胞のプレートへの接着・増殖に伴い、インピーダンスが上昇します (Hour 0-72)。

細胞がプレート上でコンフルエントになった後、低血清培地に交換し、24時間後にスクラッチ処理を施します (Hour 96)。必要に応じて評価薬剤を添加します。以降数日間に渡りCytoView-Z プレート上で細胞の遊走をラベルフリー、リアルタイムで測定します。

 

 

Maestro Z user

 

Maestro Z/ ZHT, Pro/Edgeによる細胞遊走能・浸潤能の測定:特徴

  • 経時的観察 - 細胞の遊走をリアルアルタイムで測定します。専用アプリで、実験室の外からでもライブデータの確認が可能です。

  • ラベルフリー  - 平面電極によるインピーダンス測定は、染色・試薬などを必要としません。ラベルフリー測定で数日間に渡る測定が可能です。

  • インキュベータ不要  -  Maestroには温度・CO2濃度コントローラが内蔵されています。インキュベータ等の周辺装置は不要。安定した環境下で数日間に渡る連続測定が可能です。

  • 細胞可視 - CytoView-Z 96 well プレート底面中央部は透明になっています。必要に応じて、細胞の観察が可能です。 

  • 培養から測定まで同一プレート使用 - アッセイの全行程を同一プレートで行います。他のハイスループット・プラットフォーム(例:フローサイトメータ)のような容器の入れ替えなどは不要。細胞への負担を最小限に抑えることができます。

  • スマートフォン・アプリ - 専用のスマートフォンアプリに対応しています。数日間に渡る細胞遊走の様子を、実験室の外からでも、リアルタイムに観察して頂けます。

  • 簡単 - セミ・オートメーションシステムです。ハードウエアの操作はボタン1つ。専用のソフトは、インピーダンスの変化をリアルタイムで表示します。解析結果のエクスポートも容易です。

Impedance Technology

Impedance - General

 

Impedance: For real-time cell analysis

Impedance-based cell analysis is a well-established technique for monitoring the presence, morphology, and behavior of cells in culture. Impedance describes the obstruction to alternating current flow. To measure impedance, small electrical currents are delivered to electrodes embedded in a cell culture substrate. The opposition to current flow from one electrode to another defines the impedance of the cell-electrode interface. When cells are present and attached to the substrate, they block these electrical currents and are detected as an increase in impedance.

Impedance is sensitive to many aspects of cell behavior: attachment, spreading, shape,  cell-cell connections (e.g. tight junctions), and death. Even small transient changes, such as swelling or signaling, are detectable by impedance. Because impedance is noninvasive and label free, the dynamics of these changes can be monitored in real time over minutes, hours, or even days without disturbing the biology.

Interdigitated electrodes embedded in the cell culture substrate at the bottom of each well detect small changes in the impedance of current flow caused by cell presence, attachment, and behavior.

Interdigitated electrodes embedded in the cell culture substrate at the bottom of each well detect small changes in the impedance of current flow caused by cell presence, attachment, and behavior.

In the example below, the electrodes are initially uncovered before cells are added. The electrical current passes easily and the impedance is low. When cells begin to attach and cover the electrodes, less electrical current passes and the impedance is high. After dosing with a cytotoxic agent, cells die or detach, and the impedance decreases back towards baseline.

Cells on electrode
Dosing cells and recording impedance

Impedance measures how much electrical signal (orange arrows) is blocked by the cell-electrode interface. Impedance increases as cells cover the electrode and decreases back to baseline due to cell death.

 

Continuous cell monitoring

Many cell-based assays are endpoint assays, limited to a single snapshot in time. Repeating these assays at multiple time points can be labor intensive, time consuming, and costly. Key time points can be easily missed. Impedance-based cell analysis is nondestructive and label free, meaning that cellular dynamics can be monitored continuously.

The impedance assay can be used to characterize dynamic cell profiles, revealing how cells grow, attach, and interact over time. Each cell type exhibits a different cell profile, or “fingerprint”, of dynamic cell behavior. These profiles are sensitive to cell type, density, purity, and environmental factors. In this example, the Maestro Z impedance assay readily distinguished cell profiles across different cell densities and cell types.

HeLa cells were seeded on a CytoView-Z plate at varying densities and the impedance was continuously monitored by the Maestro Z
Impedance scaled proportionally with cell density and readily distinguished different densities of the same cell type.
Maestro monitored the growth of three cell types, HeLa, A549, and Calu-3, and readily distinguishes their distinct cell profiles over time.

(A, B) HeLa cells were seeded on a CytoView-Z plate at varying densities and the impedance was continuously monitored by the Maestro Z. Impedance scaled proportionally with cell density and readily distinguished different densities of the same cell type. (C) Maestro monitored the growth of three cell types, HeLa, A549, and Calu-3, and readily distinguishes their distinct cell profiles over time.

 

 

The Maestro Z impedance assay can also be used to capture the kinetics of cell responses to drugs or immune cell therapies. The kinetics, which cannot be captured by endpoint assays, often provide key insights into the efficacy of novel therapies. In the example below, the Maestro Z impedance assay was used to quantify the kinetics of cytotoxicity of chemotherapy agents.

A549 were dosed with Doxorubicin, vehicle (DMSO), or Tergazyme. Wells dosed with Tergazyme showed an immediate decrease in impedance, reflecting complete cell death.
Cells dosed with 1 uM doxorubicin reached 50% cytolysis at 31 hrs.
Higher doses of Doxorubicin resulted in a slower decrease in impedance and cell death

A549 cells were dosed with dox, vehicle (DMSO), or tergazyme. Wells dosed with tergazyme showed an immediate decrease in impedance, reflecting complete cell death. Higher doses of dox resulted in a slower decrease in impedance and cell death. Cells dosed with 1 μM dox reached 50% cytolysis at 31 hrs.

 

Different frequencies reveal cell properties

Impedance varies with frequency, such that different frequencies reveal different aspects of cell biology. The small currents used to measure impedance will always take the path of least resistance. At low frequencies, such as 1 kHz, the impedance of the cell membrane is relatively high, forcing the current to flow under and between the cells. Low frequencies provide details about barrier integrity, the presence of gap junctions, and transepithelial or transendothelial resistance (TEER).

At high frequencies, such as 41.5 kHz, the impedance (and capacitive reactance) of the cell membrane is relatively low. Thus, most of the current couples capacitively through the cell membranes, providing information about the cell layer such as confluency and coverage.

In other words, low frequencies are sensitive to “what” cells are there, whereas high frequencies are sensitive to “how many” cells are there. The Maestro Z impedance assay uses multiple frequencies to provide the most information about the cells, simultaneously, continuously, and in real time.

Multiple frequencies were used to simultaneously and continuously monitory the coverage and barrier function
TEER, measured at 1 kHz, reveals that only Calu-3 cells form a strong barrier, as they express tight junctions to block flow between neighboring cells.

Multiple frequencies were used to simultaneously and continuously monitor the coverage and barrier function (TEER) of Calu-3 and A549 cells. Coverage, measured as resistance at 41.5 kHz, increases over time for both cell types. TEER, measured at 1 kHz, reveals that only Calu-3 cells form a strong barrier, as they express tight junctions to block flow between neighboring cells.